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Eukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species,Yarrowia lipolytica, has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion inY. lipolyticainclude codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme.

By engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and$$\alpha$$$\alpha$-amylase. In addition to secretion tag engineering, we engineered the secretory pathway by expanding the ER and co-expressing heterologous enzymes in the secretion tag processing pathway, resulting in combined 50-fold improvement in T4 lysozyme secretion.

Overall, our combined strategies not only proved effective in improving the protein production inYarrowia lipolytica, but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast.